The interactions between the vascular system and the tumor cells circulating through it play a central role in tumor metastasis. Because the molecules mediating the interactions between the circulating tumor cells and endothelium are species-specific, the use of in vivo models in rodents to study metastasis of human tumor cells can generate misleading results. We developed a novel in vitro technology, a 3-dimensional (3D) flow chamber device, which will allow researchers 1) to quantitatively measure each step of extravasation cascade of circulating tumor cells;and 2) to assess the effect of the local microenvironment on extravasation of circulating tumor cells. The objective of this proposal is to refine a design of the prototype of the 3D device with the goal to manufacture disposable and affordable 3D chambers. In Specific Aim 1, we will finalize the design of the upper compartment of the 3D device. In the first set of experiments we will optimize the pore size of the upper insert used for growing the monolayer of the endothelial cells. The optimal pore size will assure the most efficient transmigration of metastatic versus non- metastatic tumor test-cells that have circulated and subsequently adhered to the endothelial monolayer under shear stress. The second set of experiments is designed to optimize the number of wells per chamber. These experiments are required to assure that the number of transmigrated cells can be quantitatively measured to generate statistically significant results. As experimental model we propose to use the human tumor cell lines MDA-MB-435 and MDA-MB-468. In Specific Aim 2, we will test whether the 3D flow chamber device can be used to assess the effect of the local microenvironment on the adhesive interactions between circulating tumor cells and the endothelial monolayer. The goal of this Aim is to optimize the design of the lower compartment of the 3D device. Two parameters need to be adjusted to finalize the lower insert that will be used for growing cells of the local microenvironment. In the first set of experiments, we will optimize the distance between the endothelial cells and cells of microenvironment. To achieve this goal, we will manufacture and test the lower inserts with different heights of the enclosure-boundary. In the second set of experiments, we will determine the optimal pore size for the lower insert. This test is required since the optimal pore size for the upper and lower inserts is likely to be distinct from each other due to different conditions for transmigration of the tumor cells: shear stress for the upper inserts versus static conditions for the lower inserts. Furthermore, the different types of adherent cells will be cultured on the upper and lower inserts. We propose to use human MDA-MB- 435 tumor cell line as the model since these cells demonstrated high ability to adhere to endothelial cells in vivo and in vitro. Primary human lung-derived fibroblasts will be used to model local microenvironment. The studies proposed in this Phase 1 application will allow to finalize the design of the 3D device and provide proof-of-principle evidence that this device fairly represents conditions under which the circulating cells interact with vasculature in vivo. The proposed studies will lead to the development of a new commercially available in vitro technology to study molecular mechanisms that mediate migration of human tumor cells, to discover novel targets and to test drug candidates that target tumor cell migration and metastasis formation. PUBLIC HEALTH RELEVANCE: We developed a novel in vitro technology - a 3-dimensional flow chamber device - that fills a critical niche in the basic and preclinical study of tumor metastasis. This device allows to quantitatively evaluate each step of the extravasation cascade of human tumor cells under conditions of physiological shear stress. The proposed studies, when successfully completed, will lead to a new commercially available in vitro technology to study tumor cell metastasis, to discover novel targets and to test drug candidates that interfere with the process of tumor cell metastasis.